EP2021044A2 - Malleable implants containing demineralized bone matrix - Google Patents

Abstract

Described are malleable medical compositions such as pastes or putties that include solids combined with a liquid carrier. The solids can include particulate collagen and particulate demineralized bone matrix. The liquid carrier includes an aqueous medium comprising one or more polysaccharides. Also described are methods for making and using such medical compositions.

Description

MALLEABLE IMPLA N-TS CONTAINING DEMiNERALlZED BONE MATRIX

BACIvG ROUND

The present invention relates generally to medical implants. In more particular aspects, the present invention relates to osteoinductive implant formulations containing demineralized bone matrix.

A wide variety of implant formulations have been suggested in the art for the treatment of bone defects. In addition to traditional bone grafting, a number of synthetic bone graft substiαites have been used or explored, including some materials that contain demineralized bone matrix. Demineralized bone matrix has been shown to exhibit the ability to induce and/or conduct the formation of bone. It is thus desirable to implant and maintain demineralized bone matrix at a site at which bone growth is desired.

However, the beneficial nature of demineralized bone matrix is susceptible to disruption by the incorporation of incompatible materials or techniques when formulating the medical implant material At the same time, effective implant compositions must exhibit properties that retain the demineraiized bone matrix at the implant site, and that handle well in the operating environment. As well, it is of considerable commercial significance that the formulation be manufacturable without undue cost, equipment or material burdens.

In view of the background in the area of demineralized bone matrix formulations, there exist needs for product formulations which exhibit the ability to induce and/or support bone growth through a desired region, which are readily manufacturable, and which demonstrate acceptable handling properties for surgeons SUMMARY

In one aspect, the present invention provides a demineralized bone matrix composition. The composition comprises bioresorbable solids admixed with a liquid carrier to form a malleable implant composition, with the bioresorbable so! ids present at a level of about 0 1 grams per cubic centimeter (g/cc) to about 0.7 g/cc of the composition The bioresorbable solids include a particulate solid collagen materia! and a particulate solid demineralized bone matrix material, with the particulate solid collagen material present in a weight ratio no less than about i.12 and no greater than about 1 :2 relative to the particulate solid demineralized bone matrix material. The liquid carrier comprises an aqueous gel including a biocompatible polysaccharide, with the biocompatible polysaccharide present at a level of about 0.01 to about 0.5 g/cc of the composition, and in certain forms about 0.1 g/cc to about 0 5 g/cc of the composition The solid demineralized bone matrix material incorporated in the composition can exhibit osteoinductive In certain forms, such malleable compositions are tlowable paste materials in which the bioresorbable solids are present at a level of about 0.1 g/cc to about 0.25 g/cc of the composition, and the polysaccharide is present at a level of about 0.01 g/cc to about 0.03 g/cc of the composition. In other inventive embodiments, such malleable compositions are shape-retaining putties in which the bioresorable solids are present at a level of about 0 2 g/cc to about 0.4 g/cc of the composition, and the polysaccharide is present at a level of about 0.02 to about 0 5 g/cc, and in certain forms about 0 02 g/cc to about 0 05 g/cc of the composition. The polysaccharide in these inventive compositions can be an alginate, pectin or chitosan material, or a blend of any or all of these, with alginates providing particularly advantageous osteoinductive pastes and putties

In another embodiment, the invention provides a demineralized bone matrix composition that contains a liquid carrier comprising an aqueous gel including a biocompatible polysaccharide, the aqueous gel in admixture with solids to form a malleable composition. The solids of this embodiment comprise a chemically crosslinked. particulate collagen material, and a particulate demineralized bone matrix material. The demineralized bone matrix composition can exhibit osteoinductive ty In another embodiment, the invention provides a demineralized bone matrix composition thai includes a liquid carrier comprising an aqueous gel including a biocompatible polysaccharide, the aqueous gel being in admixture with particulate solids to form a malleable composition. The particulate solids include a fragmented bioresorbable sponge material and a deniineraϋzed bone matrix material. The fragmented bioresorbable sponge materia! can comprise a sponge material that has been rendered to a particulate form, such as a randomly fragmented (e.g. milled) sponge material

In another embodiment, the present invention provides a demineraϋzed bone matrix composition that includes a liquid carrier comprising an aqueous gel including a polysaccharide, the aqueous gel being in admixture with demineralized bone matrix material, wherein the demineralized bone matrix material is in the form of ribbons having a median width of at least about 0 2mm, and in certain forms at least about 0 5mm. In certain embodiments, the ribbons have a median width in the range of about 0.2mm to about 3mm (more preferably about 0 5mm to about 3mm), a median length in the range of about 5mra to about 20mm, and a median thickness in the range of about 0.02mm to about 0.2mm In certain additional embodiments, the polysaccharide is an alginate and/or the polysaccharide has a molecular weight of at least about 2 kilodaltons, e.g. in the range of about 2 to about 1000 kilodaltoπs. In further aspects, on a dry weight basis, the ribbon- form demineralized bone matrix can constitute about 70% to about 98% of the composition, more preferably about 90%^" to about 08%, and the polysaccharide can comprise about 2% to about 30% of the composition, more preferably about 10% to 2% Optionally, other insoluble solids can be included in the composition, including for example demineralized bone matrix material having other shapes and/or particulate collagen

In further embodiments, the invention provides methods of making and methods of using malleable implant compositions as described herein, and medical products including such compositions or materials for making them packaged in a sterile condition.

In still further embodiments of the invention, the demineralized bone matrix particles in any or all embodiments described herein can be characterized as, or replaced with any other, particulate collagenous material entraining one or more bone morphogenic proteins, such as BMP-2 or BMP-4. TGF~heta1 , ΪGF-1, or any combination of some or all of these growth factors.

Additional embodiments as well as features and advantages of the invention will be readily apparent to those of ordinary skill in the art from the descriptions herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. i provides a perspective view of a medical product of the invention including a medical paste or putty of the invention packaged within a terminally sterilized syringe device.

FIG. 2 provides a perspective view of a dry disruptable body that can be used to prepare malleable implant compositions of the invention

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to certain embodiments and specific language will be used to describe the same. It w ill nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated device, and such further applications of the principles of the invention as described herein being contemplated as would normally occur to one skilled in the art to which the invention relates.

As disclosed above, in certain aspects, the present invention relates to implantable medical formulations, and to methods for making and using the formulations In particular embodiments, the present invention provides medical formulations that include a malleable composition comprised of solids and a liquid carrier, such as paste or putty formulations

The solids include a substantial level of de-mineralized bone matrix particles. In particularly advantageous embodiments, the deniineraϋzed bone matrix particles will be effective to render the formulation osteoinductive when implanted in a mammalian patient, including a human patient The liquid carrier advantageous! y includes an aqueous medium containing a polysaccharide, and especially a water soluble alginate. Further, certain advantageous medical formulations include particulate collagen, such as collagen fibers or randomly fragmented collagen sponge, in an amount that is relatively less than the amount of particulate DBM on a dry weight basis. The inventive formulations desirably retain the DBM in a bioaetive, osteoinductive condition, while also providing beneficial handling and implant properties.

Malleable implant formulations of the invention will include an amount of insoluble solids combined with, the liquid carrier material. The solids include a parliculate-form demineralized bone matrix material, or another particulate-form collagenous material entraining growth factors that natively occur in bone, such as one or more bone morphogenic proteins, especially BMP-2 and/or BMP-4. transforming growth factor-beta- 1 (TGF-heta i ), insulin-like growth factor- 1 (IGF- 1 ), or any combination of some or all of these In this regard, as used herein, the term "deraineralized bone matrix" refers to a matrix material prepared by demineralizing any bone source, including cortical and/or cancellous bone. Desirable demineralized bone matrix materials will contain less than about 5% by weight of residual calcium The source bone can be from any suitable source including autogenic, allogenic, and/or xenogenic bone. When used in describing a demineralized bone matrix

(DBM) material, the term "osteoinductive" refers to the ability of the DBM material to induce bone growth Alternatively, DBM materials can be provided lacking osteoinductive character, and nonetheless be used as osteoinductive materials that provide a scaffold capable of receiving bone growth induced by natural healing processes or other materials implanted in the patient.

DBM materials for use in the present invention can be obtained commercially or can be prepared by known techniques. In general, advantageous, osteoinductive DBM materials can be prepared by decalcification of cortical and/or cancellous bone, often by acid extraction This process can be conducted so as to leave collagen, noncollagenous proteins, and growth factors together in a solid matrix. Methods for preparing such bioactive demineralized bone matrix are w el! known, in respect of which reference can be made to U.S. Patent Nos. 5,073,373; 5,484,6Oi; and 5,284,655, as examples. DBM products are also available commercially, including for instance, from sources such as Regeneration Technologies, Inc. {Atachua, FL K The American Red Cross (Arlington, VA), and others DBM materials that are solely osteoinductive can be prepared using similar techniques that have been modified or supplemented to remove or inactivate (e g. by erosslinking or otherwise denaturing) components in the bone matrix responsible for osteoinductivity Osteoinductive and/or osteoinductive DBM materials used in the present invention can desirably be derived from human donor tissue, especially in regard to implant devices intended for use in human subjects It will be understood, however, that DBM materials can also be derived from non- human animal sources and used in implants intended for use in humans or other animals.

in certain embodiments, the particulate DBM material can have an average particle size of less than about !,000 μm For instance, the DBM material can have particle sizes in the range of 50 to 850 μm.

In additional embodiments, the particulate DBM material can he in the form of elongate particles, such as fibers or ribbons. DBM ribbons having a median width of greater than about 0 5 mm are preferred, in certain embodiments with median lengths in the range of about 5 mm to about 20 mm and median thicknesses in the range of about 0 02 to about 0.2 mm Illustratively, the DBM ribbon compositions can have median widths from about 0.5 mm to about 3 mm, median lengths of about 5 mm to about 20 mm, and median thicknesses of about 0.02 to about 0,2 mm Such ribbon-form DBM particles can be made, for example, by milling off ribbons of bone from donor (e.g. human allograft) tissue, and then demineralizing the bone ribbons. Such milling can be conducted with a side-cutting bit. Alternatively or in addition, such DBM ribbons can be made by milling or grating the ribbons from a piece of demineralized cortical bone. Where ribbon-form DBM particles as described herein are used, effective formulations can be prepared which contain Sower amounts of the collagen particles, or which are even free from the collagen particles. Thus, in certain embodiments of the invention, the composition will comprise ribbon-form DBM particles as described herein, in combination with a liquid substance comprising a polysaccharide and preferably an aqueous medium comprising a polysaccharide, especially an alginate. Such compositions may comprise collagen particles or may be free from collagenous substances other than DBM The elongate ribbon form the of DBM particles promotes entanglement which, along vuih the thickening and binding nature of the polvsaceharide-coπtaining liquid carrier, can be used to provide paste or putt\ compositions of good consistency and cυhcsivencss, and in particular putty compositions with beneficial lesivtanee to deformation

In additional embodiments, the particulate DBM incorporated into the inventive composition can include a substantial component of relatively larger DBM particles in combination with relathely smaller DBM particles In certain aspects, the particulate DBM can be constituted at least 10 weight% by particles having a maximum dimension of greater than about 2 mm, or greater than about 3mm (e g in the range of about 3 mm to about 5 mm) and at least 10 weight% by particles ing a maximum dimension of less than about I mm In further aspects, the particulate DBM can be constituted at least 20 weight% b\ particles having a maximum dimension of greater than about 2mm, or greater than about 3 mm (e g in the range of about 3 mm to about 5 mm) and at least 20 weight% by particles having a maximum dimension of less than about I mm In still further embodiments, the particulate DB\1 can be constituted about 10 weigh t% to about 40 weight% b\ particles having a maximum dimension of greater than about 2mm, or greater than about 3 mm (e g in the range of about 3 mm io about 5 mm) and about 90 v\ eight% to about 60 weight°'o h\ particles having a maximum dimension of less than about 1 mm ft will be understood that particles as described above mav have the given dimensions along one axis or along two or three axes \ olumetric particles can be used, for instance having shapes ranging from generally round to generally cuboidal Products. having such DBM particle size and. or shape distributions can be prepared, for example, by blending separate DBM products ha\ ing the respective particle size distributions The presence of relathely large DBM particles in combination with smaller particles can an overall composition that resists compression upon impingement b> soft tissues at an implant site, and that also can exhibit beneficial handling and osteoinducth e properties

insoluble collagen material for use in the invention can be derived from natural tissue sources, e g \ e ogenic, allogenic, oi autogenic relatix e to the recipient human or other patient) or recombinants prepared (e g recombinant human collagen } Collagens can be subelassified into several different types depending upon their amino acid sequence, carbohvdraie content and the presence or absence of disulfide crosslinks Types I and ill collagen are two of the most common subtypes of collagen T\ pe I collagen is present in skin, tendon and bone, whereas T>pe 111 collagen is found primarily in skin The collagen used in compositions of the invention can be obtained from skin, bone, tendon, or cartilage and purified by methods well known in the art and industry Sources other than bone are preferred, in certain embodiments, for the collagen component of compositions of the invention Alternatively, the collagen can be purchased from commercial sources T\ pe 1 collagen is preferred for use in the i m ention

The collagen can be atelopeptide collagen and/or teiυpeptide collagen and can be essentially free from protein materials other than collagen Still further, either or both of non- fibrillar and fibrillar collagen can be used Non-fibrillar collagen is collagen that has been soksMlized and has not been reconstituted into its native fibrillar form

Suitable collagen products are available commercial!) , including for example from Kenscy Nash Corporation (Exton, PA), which manufactures a fibrous collagen known as Semed F, from hides Collagen materials derived from bovine hide are also manufactured b> Integra Life Science Holding Corporation (Plainsboro, NJ) Naturally - derh ed or recombinant human collagen materials are also suitable for use in the im end on illustrativ ely, recombinant human collagen products are av ailable from Kibrσgen. inc I San Fi an cisco, CA)

The solid particulate collagen incorporated into the inventive compositions can be in the form of intact or reconstituted fibers, or randomly -shaped particles, for example In certain beneficial embodiments, the solid particulate collagen will be in the form of particles deriv ed from a sponge material, foi example b> randomly fragmenting the sponge material by milling, shredding or other similar operations Such parti culafed sponge material can have an average maximum particle diameter of less than about 6 mm, more preferably less than about 3 mm, and in the range of about 0 5 mm to 2 mm Such materials can, for example, be obtained by milling oi grinding a porous sponge material and sieving the milled or ground material through a screen openings sized about b mm or smaller, desirably about 0.5 ram to about 2 mm. Retch grinders with associated sieves are suitable for these purposes. The resulting small sponge particles are randomly formed and have generally irregular shapes with remnant structures from the sponge material, and are highly beneficial for use in malleable compositions such as pastes or putties of the invention. In this regard, the use of such participated sponge materials in combination with DBM materials in malleable compositions is considered as an inventive aspect disclosed herein also wherein the sponge material is made all or in part from a bioresorbable material other than collagen. For example, the particulated sponge material can be made from any of the other natural or synthetic polymers disclosed herein. Likewise, in these particulated sponge embodiments, the liquid carrier can be a polysaccharide-coiitaining substance as disclosed herein or another suitable material, including aqueous and non-aqueous liquid mediums, and the particulated sponge material can optionally be used in the same relative amounts disclosed herein for the collagen solids materials. Further, a sponge starting material has been chemically crossϋnked with an aldehyde crossiinker such as formaldehyde or glutaraldehyde, or another suitable chemical crosslinker such as a carbodiimide. or by other techniques such as dehydroihermal or radiation-induced crosslinking, the particulated collagen or other bioresorbable materia! retains the chemical crosslinking and provides an advantageous, lasting scaffold for bone ingrowth Other sources of chemically crosslinked, particulate collagen, in fiber, irregular or other shapes, can also be used to significant advantage, and their use is considered to be another aspect of the present invention These crosslinked particulate materials can be provided as starting materials for preparing compositions as disclosed herein, and therefore as incorporated in the device these particles are individually crosslinked. As well, crosslinked solid collagen particles can be used in combination with noπ-crossiinked collagen in compositions of the invention, wherein the non-cross! inked collagen can be solid (insoluble) or soluble collagen, or combinations thereof. Such crosslinked and non-crossl inked collagen mixtures can be used, for example, to modulate the residence time of the collagen portion of the implant compositions in viva

In other advantageous embodiments, the particulate collagen, crosslinked and/or non- crosslinked, can be in the form of elongate particles, such as fibers or ribbons. Collagen ribbons having a median width of greater than about 0 2mm are preferred, more preferably greater than about 0.5 ram, in certain embodiments with median lengths in the range of about 5 mm to about 20 mm and/or median thicknesses in the range of about 0.02 mm to about 0.2 miπ Illustratively, the collagen ribbons can have median widths from about 0 2rnm to about 3mm (more preferably 0.5 rarø to about 3 mm), median lengths of about 5 mm to about 20 mm. and median thicknesses of about 0 02 mm to about 0 2 mm. When such elongate collagen ribbon compositions are used, potentially in conjunction w ith similarly-sized DBM ribbon compositions, or other DBM compositions described herein or otherwise, an advantageous mechanical entanglement of materials in the formulation can be achieved

Advantageous compositions of the invention can comprise bioresorbable solids at a level of about 0 1 grams per cubic centimeter (g/cc) to about 0.7 g/cc of the overall composition. For example, flowable paste compositions can be formulated which contain bioresorbable solids at a level of about 0.1 g/cc to about 0.25 g/cc, more typically in the range of about 0.15 g/cc to about 0 2 g'cc. Such flowable formulations can be configured as injectable materials, e g, so as to be injectable through a needle that is 16 gauge or smaller, Certain shape-retaining putty compositions can be formulated which contain bioresorbable solids at a level of about 0 2 g/cc to about 0,5 g/cc, more typically in the range of about 0,25 g/cc to about 0.35 g/cc.

As noted above, malleable formulations of certain embodiments of the invention include an amount of collagen as an insoluble solids material The collagen will desirably be incorporated in a substantial, scaffold-providing amount, but in an amount less than that of the particulate DBM on a weight-to- weight basis, hi certain compositional embodiments of the invention, the bioresorbable solids include the particulate solid collagen material and the DBM material at a collagen material. DBM material weight ratio (dry basis) of about I 12 to about 1 2. More desirably, said weight ratio is in the range of about 1 12 to about 1.5, or about I ; 10 to about 1 :5. Particular inventive embodiments are provided wherein said weight ratio is in the range of about 1 .8 to about I 5 These malleable implant compositions thus include significant amounts of the collagen solids, which provide a local scaffold material for cellular infiltration and eventual new bone volume. Typically, the particles of such collagen solids are less dense than the DBM particles, which has not only been found to improved the physical handling and implant properties of the inventive materials but can also provide a local scaffold environment that is more susceptible to cellular infiltration than the DBM particles alone. These factors, combined with the biologically-friendly nature of a thickened polysaccharide carrier, provide osteoinductive materials of particular benefit to health care providers and patients.

The liquid combined with the solids of the inventive medical formulations can be an aqueous substance, and can comprise sterile water, physiological saline, phosphate buffered saline, biood. bone marrow, bone marrow fractions or other liquid mediums, emulsions or suspensions that provide adequate wetting characteristics to form pastes, putties, and other similar malleable materials. Biocompatible organic liquids can be used in certain forms of the present invention, alone or in combination with water A wide variety of biocompatible liquids are known and suitable for these purposes, including for instance liquid polyols such as glycerol.

Compositions of the invention can be prepared in certain embodiments by mixing the insoluble solid components into the liquid material(s), preferably so as to provide a substantially homogenous composition overall. In this regard, any order of addition of the solid components may be used, As an additional preparative technique, after incorporation of the solid components into the liquid, the overall composition can be heated, desirably to a temperature that does not significantly degrade the osteoinductive character of the DBM material, when such a DBM materia! is employed. Temperatures below (>0T will be desired for these purposes, for example in the range of about 30⁰C to about 60⁰C, more preferably in the range of about 45⁰C to about 55⁰C. The heating can be conducted for any suitable period of time, in certain embodiments for a period of up to about 12 hours, and more preferably in the range of about 10 minutes to about 180 minutes. Where elongate particle forms of DBM and/or collagen are utilized in the compositions, such a heating step can be conducted so as to thicken the composition, even in the presence of the liquid carrier and in the absence of any liquid loss. Without intending to be bound by theory, it is believed that such a heating step creates a further association of the elongate DBM and/or collagen materials in the composition with one another, leading to a more viscous consistency of the malleable composition. Thus, such heating steps can be beneficial in the preparation of generally malleable or flowable compositions. The liquid phase of the medical implant compositions in accordance with certain aspects of the invention will comprise an amount of a pol) saccharide Pi ef erred are ionic polysaccharides that are capable of forming thermal!) irreversible ionicaliy-crosslinked gels upon combination with divalent or other polyvalent cationic materials Suitable such pol> saccharides include, as examples, plant-derned polysaccharides such as alginates and pectins, and gel-forming derivatives thereof Aqueous solutions of such ionic polysaccharides form iomcalh -crosslink ed gels upon contact with aqueous solutions of counter-ions For instance, useful agents for ionically crosslinking alginate and pectin poK saccharides include cationic gelling agents, preferably including divalent or trh alent cations Useful di\ alent cations for these purposes include the alkaline earth metals. especially calcium and strontium Aluminum is a useful crosslinking trivalent cation These ionic crosslinking agents will usually be provided by salts Useful anionic counter-ions for the calcium or other salts are desirably selected from pharmaceutically -acceptable anions such as chlorides, gluconates, fluorides, citrates, phosphates, tartrates, sulphates, acetates. borates, and the like An especially preferred ionic crosslinking agent for use with an alginate or pectin compound is ρro\ iάεά calcium chloride I he ionic poh saccharide chifosan can also be used (for example alone or in combination with an alginate and or a pectin), and can be ionicallv crosslinked with multh alent, anionic gelling agents Such agents include metal polyphosphates, such as an alkali metal or ammonium polyphosphate. p\ rophosphates or metaphosphates Citrates can also be used These anionic crosslinking agents will also usual!) be provided salts The cationic counter-ion for the polyphosphate or othei salt can be any suitable, biocompatible or phamiaceutically-acccptable cation including for instance sodium, potassium, or ammonium Many other biocompatible polysaccharides, including plant-derived and animal -derived materials, and standard and thickened polysaccharides, as well as corresponding ionic crosslinking agents, are known and can also be used in aspects of the present invention It will be understood that a single polysaccharide, or a blend of two or more different polysaccharides, can be used in compositions of the invention In this regard, in the case of combinations of different polysaccharides, each polysaccharide can individually be used in the amounts, ratios, ranges or levels for the polysaccharide component disclosed herein (thus potentially leading to higher values than those set forth herein when the polysaccharides arc considered on an addithe basis), or the total amount of polysaccharides < considered on an addiine basis) can be controlled to be within the amounts, ratios, ranges or levels for the polysaccharide component disclosed herein

Medical grade polysaccharides suitable for use in aspects of the invention can be prepared using known techniques or purchased from commercial sources Illustratively, purification techniques for picparing medical grade polysaccharides may include conventional separatum techniques such as chromatography, membrane filtration, precipitation, extraction, or other suitable techniques Medical grade sodium alginate ma\ be commercially obtained, for example, from MedipoS SA (Lausanne. Switzerland), or from NovaMatrix FMC Biopolyrøer (Philadelphia, Pennsylvania, Ultrapure PRONOVA brand

(endotoxin level <• 100 endotoxin units per gram))

Alginate polymers contain large variations in the total content of M and G, and the relatne content of sequence structures also varies largely (G-blocks. M-blocks and MG alternating sequences) as well as the length of the sequences along the polymer chain In some embodiments, one or more alginate polymers of the malleable composition can contain more than 50% alpha-L-guluromc acid In some embodiments, one or more alginate polymers of the composition can contain more than oO% aipha-L-guiuronic acid In some embodiments, one or more alginate polymers of the composition can contain 60% to 80% alpha-L-guluronic acid

hi certain embodiments, alginate polymers used in compositions as described herein ma\ ha\e average molecular weights ranging from 2 to 1000 kilodaltoπs (kD) The molecular weight of alginates can affect the properties of the malleable composition General!) , lower molecular weight alginates will be more biodegradable In some embodiments, the alginate polymers have an average molecule weight of from 5 to 350 kD In some embodiments, the alginate po!>roers have an average molecule weight of from 2 to 100 kD in some embodiments, the alginate polymers have an average molecule weight of from 50 to 500 kD In some embodiments, the alginate polymers have an average molecule weight of from 100 to 1000 kD I he molecular weights identified in this paragraph can similarly apply to other polysaccharides when used in the invention The alginate, when used, may possess a viscosity in a 1% solution measured at 20 degrees centigrade of from 25 to SOOO mPas and in some embodiments, 50 to 1000 mPas ( 1% solution, 20T).

While the use of those ionic polysaccharides that are capable of forming therraally- irreversible ionically-crosslinked gels is preferred, it will be understood that in the present invention these polysaccharides will be ionieally crosslinked, if at all only to an extent that does not eliminate the malleable nature of the putty, paste or other similar implant material of the present invention. Thus, within aspects of the present invention, no or substantially no ionic crossSinking agent will be added, or in some cases only a relatively small amount of ionic crosslink} ng or gelling can be added in order to increase the viscosity of the overall formulation. On the other hand, in other aspects of the invention, malleable compositions as described herein can be contacted with an amount of a liquid medium containing an ionic crosslinking agent immediately prior to, during, or after implantation of the material into a patient Illustratively, a malleable composition as described herein can be co-administered with a liquid medium of ionic crosslinker, as in the case of a dual-barrel syringe administration by which the malleable composition and crosslinker are admixed as they exit the syringe Alternatively, a previously-implanted amount of the malleable composition can be washed with a solution or other liquid medium containing an appropriate ionic crosslinking agent to stiffen the implanted materia! /w situ.

The polysaccharide will sometimes be incorporated into malleable formulations of the invention at a relatively low level. Accordingly, in certain inventive variants, the malleable implant compositions of the invention contain the polysaccharide at a level of about 0 01 g/^'cc to about 0.5 g/cc of the overall composition, sometimes about 0.01 g/cc to about 0 1 g/cc of the overall composition, and in certain forms at a level of about 0.01 g/cc to about 0.07 g/cc. In desirable flowable paste formulations, the overall composition can contain the polysaccharide at a level of about 0.01 g/cc to about 0 03 g'cc, more preferably about 0.015 to about 0 025 g/cc In certain desirable shape-retaining putty formulations, the overall composition can contain the polysaccharide at a level of about 0.02 g/cc to about 0 5 g/^'cc. hi additional embodiments, the overall composition can contain the polysaccharide at a level of about 0 02 g/cc to about 0.07 g/cc. about 0.02 g/cc to about 0 05 g/'cc, or about 0.02 g/cc to about 0.04 g/cc. It will be understood that the properties of a specific alginate composition utilized will impact its effect upon the characteristics of the overall malleable composition, and that the amount or level of alginate in inventive compositions may vary, in certain embodiments, from those specified herein

Compositions of the present invention can be manufactured in a ready-to-use format and packaged in a medically acceptable container for wetted malleable materials, in some embodiments, as illustrated in FlG I, the ready -to-use medical product can be a product 1 1 including a syringe device 12 containing an amount of a malleable composition 13 of the invention. The composition is contained within syringe barrel 14. and is transferable from the barrel 14 by actuating a plunger 15.

Compositions of the invention can also be prepared on-site, at or near the time of surgery. For instance, dry materials can be provided in the appropriate amounts, and a suitable liquid such as a buffered aqueous solution can be combined with the dry materials and mixed to form malleable implant compositions With reference to FICJ 2. in one form, ail inventive product 21 includes a body 22 of dry materials including the DBM material, collagen material, and polysaccharide is provided. Body 22 can optionally define a reservoir 23 for receiving and retaining amounts of the liquid medium to be combined with the dry materials as it soaks into the body 22. The body 22 can thereafter be disrupted, e g. by manual kneading, mixing or otherwise, to form the malleable composition.

In regard to the incorporated materia! s considered on a dry weight basis, in certain embodiments, the particulate DBM material can constitute about 40% to about 90% of the inventive compositions, more preferably about 50% to about 90%, and most preferably about

60% to about 85% by weight, In a similar vein, preferred inventive formulations can contain about 5% to about 30% by weight insoluble collagen particulate on a dry weight basis, more preferably about 8%^* to about 20%^*, and most preferably about 10% to about 15%; and can contain the polysaccharide at a level of about 1% to about 20% on a dry weight basis, more preferably about 5% to about 15%, and most preferably about 8% to about 12%. It will be understood, however, that other amounts of these materials can be used within the broader aspects of the present invention. In fuither embodiments on a din,' weight basis, ribbnn-foim DBM as described herein can constitute about 70% to about 98% of the composition, more preferabK about 90% to about 08%, and the po1> saccharide can comprise about 2% to about 30% of the composition. more preferabK about 10% to 2% In one specific embodiment, on a dr\ weight basis, the ribbon-form DBM can constitute about %% of the composition, and the polysaccharide can constitute about 4% of the composition Such compositions can be free from any collagenous material other than DBM and/or can incorporate an alginate as the polysaccharide

Further, when considered as a soivated component of the liquid phase of the composition (i e excluding the weight of the solids), the polysaccharide can for example be present as an about 1 wt% to about 4Gv, t% solution, and in certain embodiments about a 1 to about 5 vvt% solution Illustrative pastes can contain the polysaccharide in the liquid phase as an about i wt% to about 3 \\t% solution, more preferabK about 1 5 wt% to 2 5 wt% Iilustiath e, shape-retaining putties can contain the polysaccharide in the liquid phase as an about 3 \st% to 5 wt% solution, more preferabK about 3 5 \st% to 4 5 wt% Additional shape-retaining putties can contain the polysaccharide at higher levels, for example about a 5 wt% to about 40 wt% solution, in certain embodiments about a 5 ut% io about 15 v\t% solution, and in other embodiments about a 15 \vt% to about 40 wt% solution

in desirable variants, the liquid carrier of the invenine compositions is essentially or completely free from any organic compound that is a liquid at ambient or use (e g about 37 "¹C) temperatures Thus, the use of organic solvents can be avoided, and the liquid carrier in certain forms can consist or consist essentially of an aqueous solution {potentially buffered) of one or more poK saccharides that is/are effecth e to ide the desired consistency to the final formulation With respect to the admixed solids, these can be at least largely made up (e g at least about 60%, at least about 80%^". or at least about 90% by weight) by the solid collagen particles and the solid DBV! particles, and in certain embodiments the insoluble solids in the composition can consist or consist essential!) of the collagen particles and DBM particles As well, in desirable embodiments of the invention, all of the solids in the composition will be bioresorbable It will be understood, however, that these parameters are not necessary to all aspects of the invention Certain advantageous malleable compositions of the invention will contain a particulate DBM material at a level of at least about 5% by weight overall, and typically within the range of about 5% to about 30%, Such compositions can contain the particulate solid collagen material at a level of at least about 0.5% by weight overall and typically in the range of about

0 5% by weight to about 5%, and the polysaccharide at a level of at least about 0.1% by weight overall and typically in the range of about 0.1% to about 20%, and in certain forms about 0.5% to 5%. More specific inventive malleable compositions incorporate the particulate DBM material at a level of about 10% to about 25% by weight overall, the particulate solid collagen material at a level of about 1% to about 5% by weight overall, and the polysaccharide at a level of about 1% to about 20% by weight overall and in some forms about 1% to about 5% by weight overall.

The bulk densities of the inventive compositions can vary and depend upon the densities of the materials in the compositions. In certain embodiments, the bulk density of the inventive malleable composition will be in the range of about 1.1 g/cc to about 1.5 g/cc, more typically in the range of about 1.1 g/cc to about I 4 g/cc. These densities may vans however, with the densities of additional materials incorporated into the malleable compositions. For instance, bulk densities within these ranges or higher may be exhibited when amounts of dense solids such as paniculate mineralized bone or ceramics are incorporated into the formulations.

Malleable implant materials of the invention may also contain other beneficial substances including for example preservatives, cosolvents, suspending agents, viscosity enhancing agents, ionic strength and osmolality adjusters and/or other excipients. Suitable buffering agents can also be used an include but are not limited to alkaline earth metal carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates, or others Illustrative specific buffering agents include for instance sodium phosphate, sodium citrate, sodium borate, sodium acetate, sodium bicarbonate, sodium carbonate, and sodium tromethanine (TRIS). These agents can be included in amounts that are effective to maintain the pH of the system at a biologically acceptable level, for instance maintaining a pH between about 6 and about 8 and preferably near neutral The malleable implant materials disclosed heiein can also include other biocompatible and preferably bioresorbable substances These materials ma> include, for example, natural polymers such as proteins and po1> peptides, gfycosaminoglycans, proteoglycans, elastin, hyaluronic acid, dermaian sulfate, or mixtures or composites thereof poi\ niers may also be incorporated into the malleable implant materials These include, for example- biodegradable synthetic poHmers such as poϊylactic acid, poiygiyeoϋde. poh lactic polyglycolic acid copolymers ("PLGA"). poK caprolactone ("PCL"), poly(dioxanone). pol\(tri methylene carbonate) copolymers, polyglyconate, poly(propylene fumarate), poh (ethyl ene terephthalate). poly(but\ lene terephthal ate), polveth> lenegh col, polycaprolactone copolymers, polyhydroxybiuvratc. po!> hydroxy valerate, tyrosine-deriv ed polycarbonates and am random or (multi-)block copolymers, such as bϊpoh nier, terpolyrøer, quaterpolymer, etc , that can be polymerized from the monomers related to prev iously-listed homo- and copoh iners

T he malleable implant materials of the invention can also include a mineral component The mineral used can include a natural or synthetic mineral that is effective to provide a scaffold for bone ingrowth HUistraih ely, the mineral matrix mav be selected from one or more materials from the group consisting of bone particles. Bioglass\ tricalcium phosphate, bi phasic calcium phosphate, hydroxy apatite, corraline h\ droxyapatite, and biocompatible ceramics Biphasic calcium phosphate is a particular!) desirable synthetic ceramic for use in the invention Such biphasic calcium phosphate can have a tricalcium phosphate hydroxy apatite weight ratio of about 50 50 to about ^Q5 5. more preferably about 70 30 to about 95 5, even more preferably about 80 20 to about 00 10. and most preferably about 85 15 Hie mineral material can be a granular particulate having an average particle diameter between about 0 2 and 5 0 mm, more typically between about 0 4 and 3 0 mm, and desirably between about 0 4 and 2 0 mm

In another aspect of the invention, the mineral material can include bone particles, possibly cancellous but preferabh cortical, ground to pro\ ide an average particle diameter among those discussed c foi the particulate mineral material Both human and non- human sources of bone are suitable for use in the instant invention, and the bone may be autograft, allograft or xenograft in nature relative to the mammal to receive the implant Appropriate pre-treatmeπts known in the art may be used to minimize the risks of disease transmission and/or immunogenic reaction when using bone particles as or in the mineral material

In one embodiment, xenogenic bone that lias been pretreated to reduce or remove its im mutagenicity is used to provide a porous mineral matrix in the implant composition. For example, the bone can be calcined or deproteinized to reduce the risks of immunogenic reactions to the implant materia!

Bioactive agents can be delivered with malleable implant materials of the invention These bioacme agents may include, for example, antimicrobials, antibiotics, antimyobacterial, antifungals, antiviral s, antineoplastic agents, antitumor agents, agents affecting the immune response, blood calcium regulators, agents useful in glucose regulation, anticoagulants, antithrombotics, antihyperiipklemic agents, cardiac dings, thyromimetic and antithyroid drugs, adrenergics, antihypertensive agents, cholnergies, anticholinergics, antispasmodics, antiulcer agents, skeletal and smooth muscle relaxants, prostaglandins, general inhibitors of the allergic response, antihistamines, local anesthetics, analgesics, narcotic antagonists, antitussives, sedative-hypnotic agents. anticonvulsants, antipsychotics, anti-anxiety agents, antidepressant agents, anorexigenlcs, non-steroidal anti-inflammatory agents, steroidal anti-mflammaton agents, antioxidants, vasoactive agents, bone-active agents, osteogenic factors, antiarlhritics, statins, MIA/CD- RAP (melanoma-inhibiting activity/cartilage-derived retinoic acid- sensitise protein), and diagnostic agents

Bioactive agents may also be provided by tissue materials incorporated into the malleable implant material, including for instance autologous or allogenic tissue materials, which are incorporated into the material to be implanted in the patient Such tissue materials can include blood or blood fractions, bone marrow or bone marrow fractions, cartilage, and/or other sources of cells or other beneficial tissue components derived from the patient to be treated or another suitable animal source. Bioactive agents such as those described herein can be incorporated homogeneously or regionally into ihe implant materia] by simple admixture 01 otherwise Further, the\ ma> be incorporated alone or in conjunction with another carrier form or medium such as microspheres or another micropartieulate foiniulatioπ Suitable techniques for forming nήcroparticles are well known in the art, and can be used to entrain or encapsulate bioactive agents, whereafter the mieropanieles can be dispersed within the malleable implantable material upon or after its preparation

In certain embodiments, a malleable implant composition of the invention will include one or more substances, additional to the osteoinductive DBM material, that induce or generate the formation of bone Suitable osteogenic materials can include a growth factor that is effecth e in inducing formation of bone Desirably, the growth factor will be from a class of proteins known generally as bone morphogenic proteins (BMPs), and can in certain embodiments be recombinant human (rh) BMFs Ehese BMP proteins, which are known to have osteogenic, chondrυgenic and other giowth and differentiation activ ities, include rhBMP-2. rhBMP-3. rnBMP4 (also referred to as rhBMP-2B>, rhBMP- 5, rhBMP-6. rhBMP-7 (rhOP- 1), rfiBMP-8, rhBMP-9, rhBMP-12, rhBMP-13, rhBMP-J 5. rhBMP~I6. rhBMP- 17, rh BMP- 18 rtiGDF- 1 rhODF-3 rhODF-5, rhGDF-6, rhGDF-7, rhGDF-8, rhGDF-9, rhGDF-10, HiGDF- ! I , rhGDF-!2. AGDF- 14 For example, BMP-2, BMP-3, BMP-4. BMP-5, BMP-t> and BMP-7, disclosed in U S Pat Nos 5, 108,922,

5,013,640, 5, 116,738, 5,106,748, 5.187.076. and 5.141.905. BMP-8, disclosed in PCT publication WCWl - 18098. and BMP-9, disclosed in PCT publication WO93/00432, BMP- JO, disclosed in l^r S Pat No 5,637,480, BMP-U, disclosed in Ii S Pat No 5,639,638, or BMP- 12 or BMP-13. disclosed in V S Pat No 5,658.882. BMP-15, disclosed U S Pat No 5.635.372 and BMP- 16, disclosed in Il S Patent Nos 5.965.403 and 0,331,612 Other compositions which may also be useful include Vgr-2. and am of the growth and differentiation factors [GDI^'s], including those described in PCT applications WO34/15965, WO94/ 15940, WO95/0J 801, WO95/01802, WO9Φ'21681, WO94/I 5066, WO95/10539, WO96/0 I 845, WCW6/02559 and others Also useful in the present invention may be B I P, disclosed in WO94, 01557, HP0026V. disclosed in JP Publication number 7-250688, and MP52, disclosed in PCT application WO93/ 16099 The disclosures of ail of these Patents and applications are hereby incorporated herein by 2 ! reference. Abo useful in the present invention are heterodimers of the above and modified proteins or partial deletion products thereof. These proteins can be used individually or in mixtures of two or more. rhBMP-2 is preferred.

The BMP may be recombinant!}' produced, or purified from a protein composition. The

BMP may be homodimeric, or may be heterodimeric with other BMPs (e g., a heterodimer composed of one monomer each of BMP- 2 and BMP-ό) or with other members of the TGF-beta superfamϋy, such as activins, inhibins and TGF-beta 1 (e g , a heterodimer composed of one monomer each of a BMP and a related member of the TGF-beta superfamily). Examples of such heterodimeric proteins are described for example in

Published PC T Patent Application WO 93/09229, the specification of which is hereby incorporated herein by reference. The amount of osteogenic protein useful herein is that amount effective to stimulate increased osteogenic activity of infiltrating progenitor cells, and will depend upon several factors including the size and nature of the defect being treated, and the carrier and particular protein being employed

Other therapeutic growth factors or substances may also be used in malleable implant materials of the present invention, especially those that may be used to stimulate bone formation Such proteins are known and include, for example, platelet-derived growth factors, insulin-like growth factors, cartilage-derived morphogenic proteins, growth differentiation factors such as growth differentiation factor 5 (GϋF-5). and transforming growth factors, including TGF-α and TGF-β.

The osteogenic proteins or other biologically active agents, when used in the present invention, can be provided in liquid formulations, for example buffered aqueous formulations. In certain embodiments, such liquid formulations can be mixed with, received upon and/or within, or otherwise combined with a dried implant materia! containing solid ingredients in order to prepare an osteogenic, malleable implant material of the invention. One suitable rhBMP-2 formulation is available from Medtronic Sofanior Oanek. Memphis. TN, with its INFUSE® Bone Graft product Alternatively, such liquid formulations of biologically active agents can be combined with an already-prepared, netted malleable implant composition of the invention. Osteoinductive medical paste, putty or other malleable compositions of the present invention can also comprise progenitor and/or stem cells derived from embryonic or adult tissue sources and/or taken from culture. Illustratively, compositions of the invention can incorporate cells derived from blood, bone marrow , or other tissue sources from the patient to be treated (autologous cells) or from a suitable allogenic or xenogenic donor source, in certain embodiments of the invention, the conformable compositions incorporate an enriched bone marrow fraction, prepared for example as described in US Patent Publication No. 2005/0130301 to McKay et al. published June 16, 2005, publishing ILS. Patent Application Serial No. 10/887,275 filed July 8, 2004. which is hereby incorporated herein by reference in its entirety Thus, implantable materials can incorporate a bone marrow fraction enriched in connecth e tissue growth components, that is prepared by centrifuging a biological sample (e.g. from the patient to be treated) to separate the sample into fractions including a fraction rich in connective tissue growth components The fraction rich in connective tissue growth components can then be isolated from the separated sample, and incorporated into the malleable implant material, e.g by using the fraction in or as a wetting medium in combination with a dry particulate or dried body including the other ingredients of the malleable formulation

In still further embodiments, the present invention provides methods for treating patients that involve implanting in the patients a malleable implant material as described herein, in such uses, a paste, putty or other malleable implant composition of the invention can be implanted at a site at which tissue growth is desired, e.g. to treat a disease, defect or location of trauma, and/or in some instances to promote artificial arthrodesis. The medical compositions can be used as or in surgical implants at, in, on. or near bone defect sites, cartilage repair sites, or other musculoskeletal sites. Alternatively, the compositions of the invention may be applied to larger segments of bone, artificial implants, or any other kind of surgical implant. The malleable character of the compositions can enable their introduction, shaping and/or molding within voids, defects or other areas in which new tissue growth is desired, and/or in certain embodiments in which the delivery of a bioactive agent is desired. In some advantageous embodiments, the malleable material will be a putty having shape-retaining properties that desirably provide sufficient three-dimensional integrity to resist substantial compression when impinged by adjacent soft tissues of the body at a bom implant site In other advantageous embodiments, the malleable mateπai w ill he a more flowable paste, thereh) facilitating its introduction into remote implant sites using syringes, needles, tubes or other elongate delivery devices Injectable pastes, for instance effective foi injection thiough a ! 6 gauge needle, farm preferred past embodiments of the invention It will be understood, that not all pastes or putties of the invention need be injectable through reSatheS) small devices such as needles, as other delivery modes or de\ ices through open or minimally invasive surgeries wiH be available

Illustrative bone repair sties that can be treated with medical compositions of the i mention include, for instance, those resulting from injury, defects brought about during the course of surgery, infection, malignancy oi developmental malformation The malleable compositions can be used in a wide variety of orthopedic, periodontal, neurosurgical and oral and maxillofacial surgical procedures including, but not limited to the repair of simple and compound fractures and non-unions, external and internal fixations, joint reconstructions such as arthrodesis, general arthroplasty, cup arthroplasty of the hip, femoral and humeral head replacement, femoral head surface replacement and total joint replacement, repairs of the vertebral column including spinal fusion and internal fixation, tumor surger) , e g , deficit filing, discectomy, laminectomy , excision of spinal cord tumors, anterior cervical and thoracic operations, repairs of spinal injuries, scoliosis, lordosis and k\ pilosis treatments. tntermaxillar> fixation of fractures, mentoplast) , temporomandibular joint replacement, alv eolar ridge augmentation and reconstruction, inlay osteoimplanis. implant placement and icvision. sinus lifts, cosmetic enhancement, etc Specific bones which can be repaired or replaced with the malleable composition or an implant comprising the composition include, but are not limited to the ethmoid, frontal, nasal, occipital, parietal, temporal, mandible, maxilla, zygomatic, cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle, scapula, humerus. radius, ulna, carpal bones, metacarpal bones, phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella, calcaneus, tarsal and metatarsal bones

In accordance with certain aspects of the invention, the paste, putty or other malleable implant compositions of the invention can be used as bone v oid fillets, or can be incorporated in, on or around a load bearing implants such as spinal implants, hip implants (e.g. in or around implant stems and/or behind acetabular cups), knee implants {e.g. in or around stems), ϊn inventive variants, the malleable implant compositions of the invention can be incorporated in, on or around a load-bearing spinal implant device ing a compressive strength of at least about 10000 N, such as a fusion cage, dowel or other device potentially having a pocket, chamber or other cavity for containing an osteoinductive composition, and used in a spina! fusion such as an interbody fusion. One illustrative such use is in conjunction with a load-bearing interbody spinal spacer to achieve interbody fusion In these applications, the malleable implant composition can be placed in and/or around the spacer to facilitate the fusion

Illustrative cartilage repair sites that can be treated with malleable compositions of the invention include, as examples, articular cartilage surfaces occurring in articular joints having at least two major bones. Examples include, but are not limited to the elbow, wrist, phalanx, knee, and ankle. Additionally, cartilage surfaces within shoulder and hip joints can be treated.

The present invention also provides medical kits that include, or that can be used to prepare, malleable implant compositions of the invention. Such kits cars include a dried material containing the solid ingredients of the malleable formulation along with an aqueous medium or other biocompatible wetting liquid for combination with the dried material to form a malleable wetted material, or can include the formulated, wetted malleable implant material in a suitable container such as a syringe or vial Ce.g terminally sterilized), and /or another item such as a load-bearing implant (e g a spinal spacer), and/or a transfer device such as a syringe, and/or a therapeutic substance, for example an osteogenic substance such as a BMP. In one specific form, such a medical kit can include a dried material such as a particulate or dried body, a BMP in lyophilized form (e.g. rhϊϊMP-2). and an aqueous medium for reconstitute on of the BMP to prepare an aqueous formulation that can then be added to the dried material in the process of preparing an osteogenic putty, paste or other malleable implant material of the invention The invention will now be more particularly described with reference to the following specific examples. It will be understood that these examples are illustrative and not limiting of the embodiments of the invention.

Example 1

PREPARATION OF OSTEOINDUCTIVE PUTTY FORMULATION

12.9 g of sodium alginate { USP grade. Spectrum Chemical) were combined with 325 cc of phosphate buffered saline in a blender. The materials were mixed in the blender until all of the alginate was incorporated. Hie combined PBS/aiginate material was then transferred to a mixer bowl and mixing was initiated. 16 13 g of mi I led, crossHnked collagen sponge were slowly added to the mixer bowl while mixing, and the resulting materials mixed to form a uniform mixture. The milled collagen was obtained by grinding a crosslinked collagen sponge material available from Collagen Matrix, inc. (Franklin Lakes, NJ) and known as Collagen Matrix Sponge. A retch grinder operating at 8000 rpm with a i mm sieve was used for this purpose, resulting in the collection of the randomized sponge particulate as a wispy mass. 100 g of demineralized bone matrix having a particle size of 55-850 μm were then slowly added to the bowl while mixing, which was allowed to proceed until a uniform mixture was obtained. The resulting mass of putty was packed into a 60 cc syringe, and a syringe connector was attached. The 60 cc syringe was connected in sequence to smaller syringes for final packaging. In particular, 5 cc of the putty product were transferred into 6 cc syringes, and/or 10 cc of the putty product were transferred into 12 cc syringes. The top of the smaller syringe was then capped tightly, and the capped syringe was inserted into an inner pouch package which was vacuum sealed. Appropriate labels were applied to the inner pouches which were then inserted into outer packaging pouches and then heat sealed. The packaged syringe products were terminally sterilized with E-faearo to a level of 25 kiloGrays (kGy).

The osteoinductive putties prepared as above contained, on a dry weight basis, 77.5% DBM (particle size 55-850 μm), 10.0% alginate, and 12.5% collagen. On a wet weight basis, the putties contained 21.4% DBM, 2 8% sodium alginate, 3.4% collagen. and 72 4% phosphate buffered saline The compositions were of good putty quality and would retain their shape unless kneaded or pressed upon

EXAMPLE 2 PHEPARATtON OF OSTEOINDUCTIVE PASTE FORMULATION

The procedures of Example i were repeated except utilizing 660 cc of phosphate buffered saline instead of 325 cc The resulting products were of a paste character and on a dry weight basis contained the same le\els of DBM sodium alginate, and collagen as the putties prepared in Example 1 On a wet weight basis, the paste contained 12 7% DBM

I 6% sodium alginate, 2 0% collagen, and 83 7% phosphate buffered saline Hie composition had a beneficial paste consistency and was manually injectable through a I o gauge needle

KXAMPLO SPINAL FI^TSΪON STI^TDY

WITH OSTEOINDUCTIVE PASTES AND PUTTtES

hi this studs', the osteoinductive paste and putty mateπais of Examples 1 and 2 were exaJuated for their ability to induce spinal vertebral fusion when bilaterally implanted in the nude rat model

Athyraic nude rats (RNl⁵ZRKl^") ranging in weight from 212-3000 grams were obtained from Harlan Laboratories Test rats were divided into groups comprised of 6 animals according to weight Control animals received sham surgery (no test implant)

For anesthesia, animals were placed in a chamber and induced with isoflurane in oxygen (2-4%) Buprenorphine (0 5 mg/kg) was administered pre-operativcϊy for analgesia, and 0 2 ml eπrofloxacln was administered subcutaneously for antimicrobial prophylaxis prior to surgery

A dorsal midline skin incision was made from the region of L i -2 extending caudal approximate!) 3-4 cm to just caudal to the ileac crests Paramedian incisions of approximately 2 cm were made over the paraspmal fascia, and muscles were separated with a combination of blunt and shaip dissection Self-ietaining retractors were placed to retract the muscle to expose the trans\ erse vertebral processes The trans\ erse processes of the 4th and 5th lumbal \ ertebrac were identified and adherent soil tissue cleaned from the dorsal surfaces The operative sites were both flushed with approximately 1 5 ml saline Λ small, motorized burr was then used io decorticate the transverse processes io the point that bleeding bone was observed The paste or putts was then applied to the sites 0 3 ml of paste or putty compositions were applied to each site in direct contact with the decorticated transverse processes and spanned the distance between them

Surgical closure was peiibrmed in two layers using monofilament polyglycoπaie suture (4-0 J >co Healthcare) After closure, a povidone iodine ointment was placed on the wound, followed b\ application of a Tcgaderm (3Vl Healthcare) transparent dressing A radiograph was taken, and animals were placed in a sterilized polycarbonate cage with a sterile towel bed foi anesthesia recovery These cages were placed o\er a circulating warm water bath to provide supplemental heat during the recovery process \ήar several hours of observation, they were returned to single housing on it radiated bedding in saniti/ed cages Buprenorphine was administered twice daii) for two days and on the morning of the third day Enrofloxacin was administered on the e\ ening of surgery and the morning of post-operative day 1 Sutures were remox ed at approximately 7 days post-surgen , and rats were then pair housed for the study duration

The am mals on study w ere ohsen ed daily Post-operath e radiographs w ere taken 1) either immediately after surgery or at approximately i week post-surgen', 2) at approximately three weeks post-surgery and 3} at sacrifice (approximately 6 weeks post- surgery) All animal radiographs were views centered on the lower lumbar spine

Euthanasia was performed approximately 6 weeks postoperatively Animals were weighed, euthanized in a CO; chamber and radiographed The necropsy consisted of explanting the lumbar spine from 1 13 to approximate^ Sl or S2 A rib was left on 113 to aid the hisio-iechnician with site orientation F.scessive soft tissue was removed and the operatis e site identified A piece of braided suture was placed er the operain e site to aid the histo-technician with site identification The operative segment was manually palpated for motion. After the site was palpated, the spine was placed in 10% neutral buffered formalin. Fresh formaiin was exchanged 3-4 days after the necropsies were completed and the samples shipped for histopathology processing and interpretation.

The formalin fixed lumbar spines were processed (Leica TPl 050 tissue processor), embedded in paraffin, sectioned, mounted on slides and si ides were stained with hematoxylin and eosin (H&E) in an automated stainer (Leica Autostainer XL) using standard histology methodology. ϊf&E stained slides were examined by a veterinary pathologist. Coronal sections of the spines were taken to include the T4 and T5 (T -transverse processes), and the L4 and L5 (I. ^::: lumbar) vertebra Specimens were e\ aiuated for inflammatory response, which was present in all groups, and was minimal to mild in most sites. Specimens were also evaluated for the presence of fusion of T4 to T5, the presence of new bone, the presence of original DBM₅ and the presence of carrier. The results are shown in Tables 1 (Putty test subjects), 2 (Paste test subjects) and 3 (Shams), in which T ^::: Transverse process, R. ^::: Right, L ^::: Left, DBM ^::: Dernineralized bone matrix, P ~ Present, AND A ~ Absent Fusion was defined as bridging bone, with or without cartilage, between the transverse processes T4 and T5

As can be seen, bilateral new bone was observed in all six animals from in each of the paste and putty groups. Either unilateral or bilateral fusion of T4 to T5 was observed in all six animals from the putty group Unilateral fusion only (of T4 to T5) was observed in five of six animals in the paste group It should be noted that while histology provides an accurate means of evaluating bone formation, it is possible to miss bridging bone that might cross out of the plane of section under evaluation.

The spine explants described in the experimental above were also evaluated fay manual palpation after harvest The results are set forth in Table 4 below.

Table 4

Implant Fusion by Manual Palpation

Rat 1 Rat 2 Rat 3 Rat 4 fiat 5 fiat 6 Fusion

Example i Putty Yes Yes Y^τes Y^'es Y^'εs Yes 100%

Example 2 Paste Yes Yes Yes Yes Yes Yes 100% Surgical Sham No No No No No No 0%

The results demonstrate that the inventive putty and paste formulations successfully achieved fusion in the athymic rat model as assessed by manual palpation

EXAMPLE 4 EVALUATION OF OSTEOΪN DUCTI VϊTY

The athymic rat is a widely accepted animal model for testing for osteoinductive activity in DBM products This model was used to evaluate the inductivity putty formulations prepared as in Example I and paste formulations prepared as in Example 2.

The rats in the study were screened for general health before enrollment The rats were anesthetized with isoflurane throughout the surgical procedure to maintain the proper anesthetic level . Implants were placed singularly in the muscle pouch of each hind limb through a 0.5cm incision Implants were placed near the femur to allow for blood supply but not adjacent to the bone. For the putty and paste implants, 0. J cc of material was used per site. Incisions were closed with suture,

At 28 days post-operative, the rats were sacrificed via carbon dioxide inhalation The musculature wherein the sample was placed was palpated to locate the implant and discern any tangible signs of calcification All implants were harvested, preserving record of location Expiants were placed in labeled histology cassettes in 10% neutral buffered formalin.

Samples were decalcified, embedded in paraffin, sectioned & stained Three sections per implant were taken at 100 μm intervals and stained with H&E. Samples were scored semi-quantitatively according to the table below. A score of one or greater is indicative of a pass, while a score of zero is a fail for the sample. Scores are averaged to determine if the group i s a pass or fail The scoring key was as follows'

Semi-Quantitative Histologic Scoring

Score Histologic Appearance

0 No new bone form ati on

1 1 -25 % of section area is new bone and/or cartilage formation

2 25-50 % of section area is new bone and/or cartilage formation

3 50-75 % of section area is new bone and/or cartilage formation

4 75-100 % of section area is new bone and/or cartilage formation The results are set forth in Table 5 below.

Table 5 Implant Material Implant Scores

Implant 1 Implant 2 Implant 3 Implant 4 Average

Example 1 Putty Fail Pass Pass Pass Pass

Example 2 Paste Fail Pass Pass Pass Pass

Thus, the inventive putty and paste formulations were shown to be osteoinductive. EXAMPLE S

PREPARATION OF

ADDITIONAL MALLEABLE DBM FORMULATIONS

The preparative procedures described in Example 1 were used to prepare additional formulations of DBM In particular, a sodium alginate solution at a given concentration was prepared, and to this solution were added 0.3 g of collagen and 2.5 g of DBM particles. The alginates employed were a medical grade alginates with a low endotoxin level (less than 100 EU/g, UitraPure Align ate, Novamatrix, Inc.). The formulations prepared were as follows.

Table 6

Collagen tva 0.043 10.8 •-_>

DBM n/a 0.267 67.2

^♦Apparent viscosity 500 mPa's (20⁰C, \% solution) "Apparent viscosity 270 niPa's (20⁰C, 5% solution)

EXAMPLE 6

SPINAL FUSION STUBY WITH OSTEOINDUCTIVE FORMIl LATIONS

Formulations prepared as in Example 5 were used in a spinal fusion study in the nude rat mode] as generally described in Example 3, alongside a similar amount of the commercial product Acce! Connexus putty (JsoTis SA, 70% DBM in a reverse phase carrier). Microscopic examination of the right and left posterolateral sites between the transverse processes of lumbar vertebra L4 and L5 of rats from the treatment groups was conducted to determine the effectiveness of the implant formulations in promoting bone formation. Each of the formulations of Example 5 demonstrated the capacity to form bone. No significant bone formation was observed in the sham surgery. Formula 5C was observed to cause the highest average of bone formation (79%). Formula 5.B caused only slightly less bone formation (76% average). Of the remaining treatments, formula 5D caused 69% bone formation, and Formula 5 A caused 67% bone formation, on average. Accel! Connexus putty caused an average of 38% bone formation. The inventive compositions were thus demonstrated to have highly beneficial levels of osteoinductive ty useful for spinal fusion.

EXAMPLE 7

EVALUATION OF OSTEOCONDUCTIVITY IN RABBIT FEMORAL CONDYLE DEFECT STUDY

In tins example, the bone-forming capacities of four inventive formulations were evaluated and compared to the commercial product Hea!os^at bone graft material (hydroxyapatite-coated collagen microfibers) and sham surgery when implanted at a volume of about 0.15 cc's into a 5 mm diameter defect created in the cancellous bone of the distolateral femoral condyle in rabbits. The test formulations were as follows:

* Apparent viscosity 270 mPa^'s (20X, 1% solution)

15 rabbits were used in the study, to provide a total of 30 defect sites. Each test composition was implanted in 5 defect sites, randomly assigned. Six weeks after implant, tissues were harvested from the defect sites and decalcified and processed for microscopic study. Three slides were examined per each defect site. The initial evaluation was conducted in a "blinded" fashion so the treatment status of each experimental group was unknown to the pathologist. Once the evaluation was completed, the experimental groups were then "unblinded" so that the test compositions and the sham surgical procedure were properly identified prior to the reporting of the effects of each treatment Where applicable, microscopic findings were given a severity score based upon the following scale "0'" ^::: within norma! limits, " P ^::' minimal, "2" ^::: mild. "3" ^::: moderate, and "4"^::: marked. Parameters were evaluated as follows

o %Bone Formation ^:: percentage of bone seen between existent vertebra! transverse processes or percentage of test site (that may include remodeled transverse processes) that appears to be bone

o Quality of Bone ^::: percentage of bone that is either immature woven bone or mature compact bone

o %Cartilage ~ percentage of test site that contains cartilage that is undergoing transformation to bone

c Inflammation ^::: Severity rating of individual inflammatory cell types, if present, ranging from a minimal grade of "1" to a marked grade of "4" or "CT, if not present.

c Soft Tissue Reaction ^::: severity rating as previously cited of the following findings, if present, pigment, fibrosis, hemorrhage, and/or neovascularization

o Phagocytized Material ~ the presence of debris seen in the cytoplasm of macrophages

Results and Discussion.

General Observations There were a few sites that lacked both bone and soft tissue responses. The lack of findings in these instances was presumed to be due to inadequate sampling of the test sites and not treatment. As such, group averages were calculated for all sites (unadjusted) and only those sites that had some form of bone and/or soft tissue response (adjusted). Adjusted averages were viewed as a more accurate evaluation of bone formation.

Compact bone formation was by far the predominant type of bone observed with very limited amount of immature, woven bone being present. Tissue responses typically found in association with the reactive bone formation consisted mainly of macrophage and multinucleated giant cell infiltration and proliferation of connective tissue (fibrosis) Additional cellular infiltration commonly observed included heterophils and lymphocytes. Limited amounts of poorly mineralized hone fragments were at times present in areas of reactive bone. A few sites for several different treatments had fairly distinct foci of what appeared to be fluid or fibrinous accumulations.

Treatment-Related Observations. When comparing unadjusted group averages, the least amount of bone formation ( 1 1%) was found in femoral defects of the Healos^IΛ! treatment rabbits In contrast, the greatest amount of bone formation (65%) was delected in the Formula 7C treatment rabbits. Next greatest bone formation {53^O/ά} was found in the Formula 7A treatment rabbits. Comparable bone formation was detected for Surgery Sham (40%), Formula 7B (41%) and Formula 7D (46%) treatments.

When taking into account adjusted averages of bone formation, treatment-related effects were as follows Formula 7C (69%). Formula 7 A (66¹JO), Formula 7D (62% ϊ. Formula 7B (51%), Surgery Sham (50%) and Healos (23%). Based on further subjective criteria of appearance, density, and consistency of bone development the quality of bone was deemed highest for the Formula 7C and Formula 7D treatments

Lower than anticipated levels of bone formation for Formula 7B may have been due to greater variability of sampling. In addition, greater amounts of reactive bone formation may have been attributed to the surgery sham procedure during the blinded phase because of misinterpretation of existing bone due to variability in orientation of bone sections. Notwithstanding any potential erroneous bias regarding the surgery sham- induced response, there was clearly a beneficial effect of bone formation related to the inventive formulations. In summary, the microscopic examination of drill defects in the right and left femurs of 1 5 rabbits was conducted to determine the efficacy of inventive DBM implant compositions in the promotion of bone formation. This study was conducted in a blinded fashion so the actual identity of each treatment group was unknown prior to the final reporting of data. The results of this study demonstrated that the inventive DBM compositions were effective in promoting the formation of bone.

KXAMP Li: 8 PREPARATION OP BBM FORMULATION

WITH BLENDED DBM PARTICLE SIZE DISTRIBUTIONS

This example describes the preparation of a DBM putty composition with superior handling and performance upon implant, A robust, firm putty was prepared using the general procedures and ingredients described in Example 1 . The composition of the putty was;

FORMULA SA

Component g/cc overall Dry wt%

Sodium Alginate 0.38 33

Collagen 0.10 8 4

DBM 0.67 58.6

* Apparent viscosity 270 mPa^'s (2O⁰C, 1% solution)

The DBM used in this formulation was a blend of smaller and larger particle size products. Particles were first ground and screened for 2 -4mm sizes. A portion of the 2- 4mm size was reserved, and the remaining DBM was ground and screened for a 125-710 micron size. These two groups were then mixed at the time of final product manufacturing. The proportions of these two mixes were 38% 2-4mm size, and 62% 125- 7) 0 micron size. The resulting putty was cohesive and sufficiently firm to resist compression upon impingement by soft tissues at a bony implant site.

EXAMPLE 9

PREPARATION OP BBlVI FORMULATION WITH BlPHASiC CALCIUM PHOSPHATE GRANULES

This example describes the preparation of a DBM paste composition also including a significant component of granules of osteoinductive biphasic calcium phosphate (BCP) with a tri calcium phosρhate:hyc!roxyapatite ratio of about 40.60. The paste was prepared using the general procedures and ingredients described in Example 1 in addition to the BCP granules Hie composition of the material was-

The resulting paste was cohesive and contained both inorganic (ceramic granules) and organic (collagen) osteoinductive materials along with an osteoinductive DBM product

The uses of the terms "a" and "an" and "the" and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplar}' language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only preferred embodiments have been shown and described and that ail changes and modifications that come within the spirit of the invention are desired to be protected In addition, all publications cited herein are hereby incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS

1 A demineraSi/.ed bone matrix (DBM) composition, comprising bioresorbable solids admixed with a liquid carriei to form a malleable implant composition, said bioresorbable solids present at a of about 0 1 grams per cubic centimeter to about 0 7 grams per cubic centimeter of said composition. said bioresorbable solids comprising a particulate solid collagen material and a particulate solid DBM material, said particulate solid collagen material present in a weight iatio no less than about 1 12 and no greater than about 1 2 relath e io said paniculate solid DBM material, and said liquid carrier comprising an aqueous gel of a biocompatible polysaccharide, wherein said biocompatible pol\ saccharide is present at a level of about 0 01 grams per cubic centimeter to about 0 5 grams pei cubic centimeter of said composition

2 The composition of claim i, wherein said polysaccharide is selected from the group consisting of alginate, pectin and chitosan

3 The composition of claim I , wherein said polysaccharide is alginate

4 The composition of claim 1, wherein said weight ratio is' in the range of I 12 to 1 5

5 The composition of claim 1 , w herein said weight ratio is in the range of

I 10 to 1 5

e> The composition of claim i, wherein said bioresorbable solids are present at a level of about 0 1 g/cc to about 0 25 g cc of said composition, said polysaccharide is present at a level of about 0 01 g''cc to about 0 03 g cc of said composition, and 4 ! said composition is a flowable paste

7 The composition of claim I, wherein said bioresorable so! ids arc present at a c! of about 0 2 g/cc to about 0 4 g/cc of said composition, said poi) saccharide is present at a Ie\ e! of about 0 01 g/cc to about 0 5 g<ec of said composition, and said composition is a shape-retaining putty

8 Hie composition of claim 6, w herd n said poK saccharide i s an aigi nate

^c) The composition of claim 7, wherein said polysaccharide is an alginate

10 The composition of claim 8, wherein said % eight ratio of collagen material to DBM material is in the range of I 10 to I 5

i 1 The composition of claim % wherein said weight ratio of collagen material to DBM material is in the range of 1 10 to 1 5

12 The composition of claim 1 , wherein said particulate solid collagen material comprises a chemicalK crosslinked collagen material

13 The composition of claim ! 2, wherein said particulate solid collagen material comprises a partial! a ted sponge material

54 The composition of claim !. also comprising a mineral material selected from synthetic ceramic granules or roineiali/ed bone chips

15 The composition of claim 1, wherein said solid DBM material in said composition exhibits osteoinducthity 16 A demineralized bone matrix (DBM) composition, comprising' a Ijqiiid carrier comprising an aqueous gel of a biocompatible polysaccharide, said aqueous gel in admixture with solids to form a malleable composition, said solids comprising a chemically crossiinked, particulate collagen material; and said solids comprising a particulate DBM material.

17. The DBM composition of claim 16, wherein said polysaccharide is selected from the group consisting of alginate, pectin, and chitosan.

18. The DBM composition of claim 16. wherein said polysaccharide is an alginate.

19. The DBM composition of claim 16, wherein said particulate DBM material is osteoinductive

20. The DBM composition of claim 1°₅ wherein said participated sponge material has an average particle size less than about 2 ram.

21. The DBM composition of claim 16, wherein said particulate collagen material is present in a weight ratio no less than about 1 : 12 and no greater than about i.2 relative to said particulate DfJM material

22. The DBM composition of claim 2L wherein said weight ratio of particulate collagen material to particulate DBM material is in the range of 1 : 10 to 1 :5.

23. The DBM composition of claim 21, wherein said polysaccharide is an alginate

24 The DBM composition of claim 16, wherein: said solids are present at a level of about 0 1 g/cc to about 0.25 g/cc of said composition; said poh saccharide is present at a level of about 0 01 g/cc to about 0 03 g/cc of said Ct)R) position, and said composition is a flowable paste

25 ϋie DBM composition of claim ! o. wherein said solids are present ai a level of about 0 2 g'cc to about 0 4 g/cc of said composition. said polysaccharide is present at a level of about 0 01 g/cc to about 0 5 g/cc of said composition, and said composition is a shape-retaining putty

26 Λ method for treating a patient, comprising implanting an amount of a composition according to claim i in the patient at a location at which bone growth is desired

27 Λ method for making a deminerali/ed bone matrix (DfJM) composition, the method comprising forming an admixture including bioresorbable solids and a liquid carrier, said bioresorbable solids present at a level of about 0 1 grams per cubic centimeter to about 0 7 grams per cubic centimeter of said composition. wherein said bioresorbable solids comprise a particulate solid collagen material and a particulate solid DBM mateiiai. said paniculate solid collagen material present in a weight ratio no 3ess than about 1 12 and no greater than about 1 2 relative to said particulate solid DBM material, and wherein said liquid carrier comprises an aqueous gel of a biocompatible polysaccharide, with said biocompatible polysaccharide present at a level of about 0 01 grams per cubic centimeter to about 0 5 grams per cubic ccntimetei of said composition

28 \ method for making a demineralized bone matrix (DBM ) composition, the method comprising forming an admivtuie including a liquid carrier and solids, said liquid carrier comprising an aqueous ge! of a biocompatible po!> saccharide. wherein said solids comprise a chemicalh crosslmked, particulate collagen material, and wherein said solids comprise a particulate DBM material

29 A deminerali/ed hone matrix (DBM) composition, comprising a liquid carrier comprising an aqueous gel including a biocompatible polysaccharide, said aqueous gel in admixture with particulate solids to form a malleable composition. said particulate so! ids comprising a participated bioresorbable sponge material, and said particulate solids comprising a DBM material

30 The DBM composition of claim 37, wherein said participated bioresorbable sponge material comprises collagen

31 The DBM composition of claim 37, wheiom said polysaccharide is selected from the group consisting of alginate, pectin and chitosan

32 The DBM composition of claim 39 wherein said pol) saccharide is an alginate

33 The DBM composition of claim 37, wherein said sponge material is chemically crυsslmked

34 The DBM composition of claim 37, wherein said DBVl material is osteoinductive

35 A method for preparing a dcmirteraii/ed bone matrix (DBM) composition, comprising admixing particulate solids with a liquid carrier comprising an aqueous gel including a biocompatible poh saccharide to form a malleable composition, wherein said particulate solids comprise a particulate*! bioresorbable sponge material, and wherein said particulate solids comprise a DBM material.

36. The composition of claim 1, wherein said particulate solid DBM material includes elongate particles having a width of at least about 0.2 mm.

37 The DBM composition of claim 16, wherein said particulate solid DBM material includes elongate particles having a width of at least about 0.2 mm.

38. The DBM composition of claim 37, wherein said particulate solid DBM material includes elongate particles having a width of at least about 0.2 mm.